1. Field of the Invention
This invention relates to a method of assessing oxidative stress-in vivo by quantification of markers and their metabolites formed by free radical mediated oxidation.
2. Description of Related Art
Free radicals derived primarily from oxygen have been implicated in the pathophysiology of a number of human diseases, such as atherosclerosis, ischemia-reperfusion injury, inflammatory diseases, cancer and aging. A variety of methods have been developed to assess oxidative stress; however, some of these methods have limited sensitivity or specificity, while others are either too invasive or not adaptable for human investigation. Halliwell, B., et al. The Measurement Of Free Radical Reactions In Humans: Some Thoughts For Future Experimentation. FEBS Letters. 213:9-14, 1987.
Unfortunately, oxidative stress is difficult to assess in humans due to lack of reliable methods to assess oxidant stress in vivo. As one author stated, xe2x80x9cone of the greatest needs in the field now is the availability of a non-invasive test to probe the is oxidative stress status of humans.xe2x80x9d Id.
Regional increases in oxidative damage are a feature of brain tissue obtained post mortem from patients with Alzheimer""s disease (reviewed in Markesbery, W. R., 1997). However, an objective index of oxidative damage associated with AD that may be assessed during life is lacking. Such a biomarker could have an important impact on the ability to test hypotheses concerning oxidative damage in AD patients by permitting repeated evaluation to follow progression of disease and to quantify response to experimental therapeutic interventions.
Lipid peroxidation is a prominent manifestation of oxidative challenge in brain (reviewed in Markesbery, WR, 1997). Recently, it has been shown that markers of lipid peroxidation are increased in cerebrospinal fluid (CSF) of AD patients compared to control subjects (Lovell et al, 1997; Montine et al., 1997). Although these studies suggest that quantification of lipid peroxidation products in CSF may provide an intra vitam index of oxidative damage to brain, the assays employed have shortcomings, including the need for large volumes of CSF and measuring highly reactive molecules, such as 4-hydroxynonenal, that limit their interpretation or widespread application.
Previously, a series of prostaglandin F2-like compounds, termed F2-isoprostanes (F2-IsoPs), were disclosed that are produced by free radical-catalyzed peroxidation of arachidonic acid independent of the cyclooxygenase enzyme (Morrow et al., 1990). Significant advantages to quantifying F2-IsoP as an index of oxidative stress are their specificity for lipid peroxidation, their chemical stability, and the relatively small tissue volumes required for their detection.
Free radicals are generally short lived and thus, indirect methods of detection are required. Pryor, W., On The Detection Of Lipid Hydroperoxides In Biological Samples, FREE RADICAL BIOLOGY and MEDICINE, Vol. 7, pages 177-178, 1989. Standard detection methods include: electron spin resonance (directly), electron spin resonance (spin trapping), thiobarbituric acid reactive substances (TBARS), detection of malonaldehyde by direct methods (such as HPLC of malonaldehyde itself or as its dinitrophenylhydrazone), detection of other oxidation products from polyunsaturated fatty acids (such as 4-hydroxynonenal), measurement of lipid hydroperoxides, detection of volatile hydrocarbons (ethane, pentane and ethylene), detection of oxidation products from lipids other than polyunsaturated fatty acids (e.g., cholesterol), oxidation of methional, methionine, or 2-keto-4-thiomethylbutanoic acid to ethylene, oxidation of benzoic acid to carbon dioxide (often with radiolabelled carbon dioxide), oxidation of phenol benzoic acid, or aspirin to hydroxylated products, determination of decreases in antioxidant levels (e.g., decreased GSH, tocopherol, or ascorbate) or of increases in the oxidized products from antioxidants (e.g., tocopherol quinone or the ascorbyl radical), detection of oxidized DNA bases (e.g., thymine glycol, 8-hydroxydeoxyguanosine), detection of oxidized products from proteins (e.g., methionine sulfoxide from methionine) or of proteins oxidized to carbonyl-containing products that then react with hydride-reducing agents, detection of adducts of DNA bases (e.g., by enzymatic hydrolysis post-labeling using P32), and chemi-luminescence methods. Id.
Also, docosahexaenoic acid (C22:6xcfx893)(DHA) has been the subject of considerable interest owing to the fact that it is highly enriched in the brain, particularly in gray matter, where it comprises approximately 25-35% of the total fatty acids in aminophospholipids (Salem et al., 1986; Skinner et al., 1993). Although DHA is present in high concentrations in neurons, neurons are incapable of elongating and desaturating essential fatty acids to form DRA. Rather, DHA is synthesized primarily by astrocytes after which it is secreted and taken up by neurons (Moore et al., 1991). Although the precise function of DHA in the brain is not well understood, deficiency of DHA is associated with abnormalities in brain function (Conner et al., 1992). Applicants considered the possibility that IsoP-like compounds could be formed by free radical-induced peroxidation of DHA. Because such compounds would be two carbons longer in length than IsoPs, it would be inappropriate to term these compounds IsoPs. Since DHA is highly enriched in neurons in the brain, applicants therefore propose to term these compounds xe2x80x9cneuroprostanesxe2x80x9d (NPs).
It would therefore be useful to develop additional methods for assessing oxidative stress in vivo which are neither too invasive nor limited to animal models.
According to the present invention, there is provided a method of assessing oxidative stress in vivo by measuring an amount of neuroprostanes in a biological sample before the ex vivo development of neuroprostanes in a sample, comparing the measured amount of neuroprostanes with a control and assessing oxidative stress in vivo based on this comparison. There is also provided a marker for oxidative stress by the increase of neuroprostanes in a biological sample compared to a control sample. A diagnostic tool for determining the presence of a neurodegenerative disease determines an increased amount of neuroprostanes in a biological sample compared to that of a control sample.